1. Field of the Invention
The present invention relates to a method for detecting the presence of bacteria in a sample using fluorescent conjugates. The present invention also relates to a method for detecting the presence of bacteria in a sample by measuring the fluorescent intensity ratio of a metabolized fluorescent product at a specific wavelength to the metabolizable fluorescent conjugate at a second specific wavelength.
2. Description of the Art
Bacterial contamination of products designed for human use or consumption can be a significant public health and public relations problem. Recent news reports in this country and abroad have alerted the public to the dangers of bacterial contamination. As a result, rapid methods to identify sources of bacterial contamination have become increasingly important.
The sources of bacterial contamination frequently arise from inoculation of pathogenic bacteria into water sources via intestinal excrement of animals, particularly mammals. Epidemiological studies have indicated a strong correlation between the presence of waterborne pathogenic organisms and the presence of microorganisms of mammalian intestinal origin (e.g. the bacterium Esherichia coli). In addition, it has been well established that the quality of products for human use or consumption is negatively impacted by the presence of pathogenic organisms. Coliform bacteria (e.g., bacteria normally present in the gut of humans or animals) in particular have been shown to correlate with outbreaks of certain diseases such as gastroenteritis, dysentery, hepatitis, and the like. These illnesses can be severe or life-threatening in children, the elderly, and immunosuppressed individuals. Bathing beaches, shellfish beds, food processing and/or handling facilities, foodstuffs (e.g., hamburger, chicken, apple cider and the like), hospital equipment, sewage treatment plants, and certain industrial lubricants are a few examples of products or locations that can become contaminated by coliform bacteria. If present in significant numbers, coliform bacteria can cause product deterioration, spoilage, disease, or serious economic loss of consumable goods. In addition, the public perception about the sanative state of a product or location can be damaged, and is difficult to repair. It is therefore important to identify those products or locations that are contaminated with coliform bacteria as quickly as possible.
To address the problem of bacterial contamination, several test methodologies have been developed. Among these are radiometric, electrochemical, chromatographic, chemiluminescent, and fluorescent methodologies.
Radiometric approaches to identifying bacterial contamination generally utilize a nutrient such as lactose or galactose that incorporates one or more radioactive atoms. Upon metabolization by the bacteria, the radiolabel becomes incorporated into the bacteria, and the bacteria can be isolated and identified by following the radiolabel. However, this method has several undesirable drawbacks. Although this method is very sensitive, it utilizes radioisotopes which can be expensive and difficult to handle. This method also generates significant quantities of radioactive waste which is difficult and expensive to dispose. In addition, testing procedures using this method generally require from about 18 to 48 hours to produce results. This time delay in identifying the source of the bacterial contamination is too great in many situations that require immediate identification.
Electrochemical, chromatographic, and chemiluminescent methods are generally less sensitive than the radiometric approaches, and therefore require higher concentrations of bacteria to generate useful data. To achieve these higher bacterial concentrations, the bacteria are frequently cultured in a growth medium for about 24 hours until enough bacteria are present for the test. Alternatively, the bacteria are concentrated using filtration or other concentrating procedures. However, these preliminary concentration steps require additional time, materials, and equipment which generally add complexity to these procedures. In addition, the total time for performing electrochemical, chromatographic, and chemiluminescent tests generally range from 24 to about 48 hours. Like the radiometric procedures, this time delay may be too great if information concerning bacterial contamination is urgently needed.
Several fluorescent methods have been described in the prior art which offer advantages over the radiometric, electrochemical, chromatographic, and chemiluminescent methods. Many of these fluorescent methods are based on the enzymatic degredation of a fluorescently labeled umbelliferone substrate with concomitant monitoring of the fluorescence at a single wavelength. The following U.S. patents are representative of the current state of the art:
U.S. Pat. No. 4,591,554 to Koumura et al. discloses a method for rapidly detecting microorganisms utilizing nonfluorescent umbelliferone derivatives such as 4-methyl-umbelliferyl-.beta.-D-galactoside, 4-methyl umbelliferyl-.alpha.-D-galactoside, 4-methyl umbelliferyl-phosphate, and 4-methyl umbelliferyl-pyrophosphate. Fluorescence of the liberated umbelliferone moiety is induced at 360 nm and monitored at 450 nm. High concentrations of umbelliferone derivative are used in this method (10.sup.-3 -10.sup.-4 M).
U.S. Pat. No. 5,089,395 to Snyder et al. discloses use of a nonfluorescent umbelliferone derivative which is enzymatically converted to a fluorescent product to indicate the presence of bacteria. High concentrations of umbelliferone derivative are used in this method (about 50 .mu.g/ml). Fluorescence of diacetylfluorescein is induced at 310 nm and monitored at 350 nm to calibrate the instrument.
U.S. Pat. No. 5,518,894 to Berg discloses a rapid method to detect the presence of coliform bacteria. However, this method uses a concentration step (filtration) in combination with incubation to increase the number of bacteria present. The fluorescence of a high concentration of hydrolyzed umbelliferone derivative is monitored as an indication of bacterial presence. Fluorescence is induced at 365 nm and monitored at 465 nm.
U.S. Pat. No. 5,429,933 to Edberg discloses detection of environmental microbes using a fluorescent metabolite and a nutrient-rich medium. No particular wavelengths to monitor fluorescence emission energy are disclosed.
U.S. Pat. No. 5,610,029 to Ehrenfeld et al. discloses a culture medium for the detection of microbes in a sample. This culture medium includes various nutrients and growth factors, as well as a fluorescent metabolite (4-methyl umbelliferyl-.beta.-D-glucuronide). High concentrations of the fluorescent metabolite are utilized (60-90 .mu.g/ml), and fluorescence is monitored at 366 nm.
Thus, many of the prior art methods of detecting bacteria in a sample utilize high concentrations of fluorescent metabolite, and monitor induced fluorescence at a single wavelength. However, it is well established that high concentrations of fluorescent compounds result in a quenching effect of induced fluorescence. Thus, an accurate correlation between induced fluorescence and bacterial presence is not possible using high concentrations of fluorescent metabolite. Additionally, monitoring fluorescence with a single wavelength requires instrument and temperature stability, as well as calibration of the instrument prior to each experiment. Such requirements make the analysis process time-consuming, unreliable, and highly dependent on operator skill. Accordingly, what is needed in the art is a rapid method to detect the presence of bacteria in a sample that is simple to perform, accurate, and reliable. The present invention is believed to be an answer to that need.